We evaluated the feasibility of same-day routine aerobic bacterial identification using the following procedures: Picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The matched identification rate of this procedure at the species level was 80.6% (141/175) for the 4-h cultures compared with overnight cultures and 90.9% (159/175) for the 6-h cultures. Thus, our technique provides an easy and rapid method for identification of aerobic bacteria in routine clinical microbiology laboratories.